SUMMARY BACKGROUND DATA:

Small interfering RNA (siRNA) molecules suppress expression of target genes and may have therapeutic applications as target-specific therapies for cancer. Therefore, the purpose of this study was 2-fold: 1) to analyze the distribution pattern of PI3K pathway components in human normal colorectal cancers, and 2) to determine whether targeted inhibition of PI3K inhibits colon cancer growth in vitro and suppresses metastatic growth in vivo.

METHODS:

Immunohistochemical analysis was performed on colorectal adenocarcinomas and adjacent normal mucosa for PI3K pathway components, including p85alpha, p110alpha, Akt1, Akt2, and the tumor suppressor PTEN, which inhibits PI3K. HT29 and KM20 human colon cancer cells were treated with siRNA directed to p85alpha or p110alpha, and cell viability and apoptosis assessed. HT29 cells, transfected with a plasmid containing green fluorescent protein (GFP), were injected into the spleen of athymic nude mice to establish liver metastases; mice were randomized to receive either nontargeting control (NTC), p85alpha or p110alpha siRNA.

RESULTS:

PI3K pathway components p85alpha and Akt2 were highly expressed in glandular elements of colon cancers, with a correlation between staining intensity and clinical stage; PTEN expression was decreased in the colon cancers of all stages. PI3K-specific siRNA treatment decreased cell viability in vitro and suppressed metastatic tumor growth in vivo.

FIGURE 2. siRNA directed against p85α or p110α inhibits proliferation. The effect of siRNA directed to PI3K components on the viability of KM20 (A) or HT29 (B) cells was assessed. Cell viability was measured as described in Materials and Methods. Points represent means of triplicate determinations ± SD. *P < 0.05 for p85α, p110α siRNA compared with nontargeting control (NTC) siRNA. KM20 (C) or HT29 (D) cells transfected with p85α, p110α, or NTC. siRNA sequences were lysed and Western blots performed using anti-Akt, phospho (Ser473), anti-p85α, and anti-p110α; β-actin was used as a loading control (bottom row).

FIGURE 4. Suppression of metastatic tumor growth by p85α or p110α siRNA. A, HT29-GFP cells (5 × 106) were inoculated intrasplenically and mice were killed 5 weeks later. Animals were monitored individually for metastatic tumor growth using the Illumatool TLS. Representative images of nude mice at the end of the experiment on day 35 are shown. B, Animals were randomized into 3 experimental groups (5 animals per group) to receive p85α, p110α, or nontargeting siSTABLE siRNA (20 μg/mice, qod) by hydrodynamic tail vein injection 24 hours after intrasplenic injection; mice were killed 35 days later. Using Adobe Photoshop, the level of fluorescence was measured and expressed as a pixel number. All tests were assessed at the 0.05 level of significance. C, Representative images of nude mouse livers at the end of the experiment on day 35.